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1.
Cell Rep ; 41(7): 111670, 2022 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-36384122

RESUMO

In healthy vessels, endothelial cells maintain a stable, differentiated, and growth-arrested phenotype for years. Upon injury, a rapid phenotypic switch facilitates proliferation to restore tissue perfusion. Here we report the identification of the endothelial cell-enriched long non-coding RNA (lncRNA) PCAT19, which contributes to the proliferative switch and acts as a safeguard for the endothelial genome. PCAT19 is enriched in confluent, quiescent endothelial cells and binds to the full replication protein A (RPA) complex in a DNA damage- and cell-cycle-related manner. Our results suggest that PCAT19 limits the phosphorylation of RPA2, primarily on the serine 33 (S33) residue, and thereby facilitates an appropriate DNA damage response while slowing cell cycle progression. Reduction in PCAT19 levels in response to either loss of cell contacts or knockdown promotes endothelial proliferation and angiogenesis. Collectively, PCAT19 acts as a dynamic guardian of the endothelial genome and facilitates rapid switching from quiescence to proliferation.


Assuntos
RNA Longo não Codificante , Fosforilação , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Células Endoteliais/metabolismo , DNA/metabolismo , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo
2.
Sci Rep ; 10(1): 19079, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33154469

RESUMO

Zinc finger proteins (ZNF) are a large group of transcription factors with diverse functions. We recently discovered that endothelial cells harbour a specific mechanism to limit the action of ZNF354C, whose function in endothelial cells is unknown. Given that ZNF354C has so far only been studied in bone and tumour, its function was determined in endothelial cells. ZNF354C is expressed in vascular cells and localises to the nucleus and cytoplasm. Overexpression of ZNF354C in human endothelial cells results in a marked inhibition of endothelial sprouting. RNA-sequencing of human microvascular endothelial cells with and without overexpression of ZNF354C revealed that the protein is a potent transcriptional repressor. ZNF354C contains an active KRAB domain which mediates this suppression as shown by mutagenesis analysis. ZNF354C interacts with dsDNA, TRIM28 and histones, as observed by proximity ligation and immunoprecipitation. Moreover, chromatin immunoprecipitation revealed that the ZNF binds to specific endothelial-relevant target-gene promoters. ZNF354C suppresses these genes as shown by CRISPR/Cas knockout and RNAi. Inhibition of endothelial sprouting by ZNF354C is dependent on the amino acids DV and MLE of the KRAB domain. These results demonstrate that ZNF354C is a repressive transcription factor which acts through a KRAB domain to inhibit endothelial angiogenic sprouting.


Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Neovascularização Fisiológica , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Sistemas CRISPR-Cas , Células Cultivadas , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Histonas/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Mutagênese Sítio-Dirigida , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Neovascularização Fisiológica/genética , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteína 28 com Motivo Tripartido/metabolismo , Dedos de Zinco/genética
3.
Cardiovasc Res ; 115(14): 1963-1974, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-30949676

RESUMO

AIMS: The protein Scrib (Scribble 1) is known to control apico-basal polarity in epithelial cells. The role of polarity proteins in the vascular system remains poorly characterized; however, we previously reported that Scrib maintains the endothelial phenotype and directed migration. On this basis, we hypothesized that Scrib has anti-atherosclerotic functions. METHODS AND RESULTS: Tamoxifen-induced Scrib-knockout mice were crossed with ApoE-/- knockout mice and spontaneous atherosclerosis under high-fat diet (HFD), as well as accelerated atherosclerosis in response to partial carotid artery ligation and HFD, was induced. Deletion of Scrib resulted in increased atherosclerosis development in both models. Mechanistically, flow- as well as acetylcholine-induced endothelium-dependent relaxation and AKT phosphorylation was reduced by deletion of Scrib, whereas vascular permeability and leucocyte extravasation were increased after Scrib knockout. Scrib immune pull down in primary carotid endothelial cells and mass spectrometry identified Arhgef7 (Rho Guanine Nucleotide Exchange Factor 7, ßPix) as interaction partner. Scrib or Arhgef7 down-regulation by siRNA reduced the endothelial barrier function in human umbilical vein endothelial cells. Gene expression analysis from murine samples and from human biobank material of carotid endarterectomies indicated that loss of Scrib resulted in endothelial dedifferentiation with a decreased expression of endothelial signature genes. CONCLUSIONS: By maintaining a quiescent endothelial phenotype, the polarity protein Scrib elicits anti-atherosclerotic functions.


Assuntos
Aterosclerose/prevenção & controle , Polaridade Celular , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Permeabilidade Capilar , Adesão Celular , Movimento Celular , Polaridade Celular/genética , Modelos Animais de Doenças , Células Endoteliais/patologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Fenótipo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais , Transcriptoma , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Vasodilatação
4.
Theranostics ; 8(8): 2117-2133, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29721067

RESUMO

RATIONALE: Classic histology is the gold standard for vascular network imaging and analysis. The method however is laborious and prone to artefacts. Here, the suitability of ultramicroscopy (UM) and micro-computed tomography (CT) was studied to establish potential alternatives to histology. METHODS: The vasculature of murine organs (kidney, heart and atherosclerotic carotid arteries) was visualized using conventional 2D microscopy, 3D light sheet ultramicroscopy (UM) and micro-CT. Moreover, spheroid-based human endothelial cell vessel formation in mice was quantified. Fluorescently labeled Isolectin GS-IB4 A647 was used for in vivo labeling of vasculature for UM analysis, and analyses were performed ex vivo after sample preparation. For CT imaging, animals were perfused postmortem with radiopaque contrast agent. RESULTS: Using UM imaging, 3D vascular network information could be obtained in samples of animals receiving in vivo injection of the fluorescently labeled Isolectin GS-IB4. Resolution was sufficient to measure single endothelial cell integration into capillaries in the spheroid-based matrigel plug assay. Because of the selective staining of the endothelium, imaging of larger vessels yielded less favorable results. Using micro-CT or even nano-CT, imaging of capillaries was impossible due to insufficient X-ray absorption and thus insufficient signal-to-noise ratio. Identification of lumen in murine arteries using micro-CT was in contrast superior to UM. CONCLUSION: UM and micro-CT are two complementary techniques. Whereas UM is ideal for imaging and especially quantifying capillary networks and arterioles, larger vascular structures are easier and faster to quantify and visualize using micro-CT. 3D information of both techniques is superior to 2D histology. UM and micro-CT together may open a new field of clinical pathology diagnosis.


Assuntos
Vasos Sanguíneos/diagnóstico por imagem , Imageamento Tridimensional , Microscopia/métodos , Microtomografia por Raio-X , Animais , Colágeno/farmacologia , Vasos Coronários/diagnóstico por imagem , Combinação de Medicamentos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Rim/irrigação sanguínea , Laminina/farmacologia , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica , Placa Aterosclerótica/diagnóstico por imagem , Placa Aterosclerótica/patologia , Proteoglicanas/farmacologia
5.
Arterioscler Thromb Vasc Biol ; 35(9): 1954-62, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26205961

RESUMO

OBJECTIVE: The polarity protein Scrib is highly expressed in endothelial cells and is required for planar cell polarity. Scrib also facilitates recycling of integrin α5 to the plasma membrane. Because integrin α5 signals the presence of the inflammatory matrix protein fibronectin, we hypothesized that Scrib contributes to endothelial inflammatory signaling. APPROACH AND RESULTS: Cytokine treatment of human umbilical vein endothelial cells induced an inflammatory response as evident by the induction of vascular cell adhesion molecule-1 (VCAM-1). Downregulation of Scrib greatly attenuated this effect. In endothelial-specific conditional Scrib knockout mice, in vivo lipopolysaccharide treatment resulted in an impaired VCAM-1 induction. These effects were functionally relevant because Scrib small interfering RNAs in human umbilical vein endothelial cells attenuated the VCAM-1-mediated leukocyte adhesion in response to tumor necrosis factor-α. In vivo, tamoxifen-induced endothelial-specific deletion of Scrib resulted in a reduced VCAM-1-mediated leukocyte adhesion in response to tumor necrosis factor-α in the mouse cremaster model. This effect was specific for Scrib and not mediated by other polarity proteins. Moreover, it did not involve integrin α5 or classic pathways supporting inflammatory signaling, such as nuclear factor κ light chain enhancer of activated B-cells or MAP kinases. Co-immunoprecipitation/mass spectrometry identified the zinc finger transcription factor GATA-like protein-1 as a novel Scrib interacting protein. Small interfering RNA depletion of GATA-like protein-1 decreased the tumor necrosis factor-α-stimulated VCAM-1 induction to a similar extent as loss of Scrib did. Silencing of Scrib reduced GATA-like protein-1 protein, but not mRNA abundance. CONCLUSIONS: Scrib is a novel proinflammatory regulator in endothelial cells, which maintains the protein expression of GATA-like protein-1.


Assuntos
Artérias Carótidas/metabolismo , Fator de Transcrição GATA1/genética , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Inflamação/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , RNA/genética , Animais , Western Blotting , Artérias Carótidas/patologia , Células Cultivadas , Modelos Animais de Doenças , Fator de Transcrição GATA1/biossíntese , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais
6.
Arterioscler Thromb Vasc Biol ; 27(8): 1736-43, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17541028

RESUMO

OBJECTIVE: Basic fibroblast growth factor (bFGF) stimulates vascular smooth muscle cell (SMC) migration. We determined whether bFGF increases SMC reactive oxygen-species (ROS) and studied the role of ROS for SMC migration. METHODS AND RESULTS: bFGF rapidly increased rat SMC ROS formation and migration through pathways sensitive to inhibition of NADPH oxidases, PI3-kinase, protein kinase C, and Rac-1. SiRNA directed against the NADPH oxidase Nox4 impaired basal but not bFGF-induced ROS formation and did not affect migration. In contrast, siRNA against Nox1 blocked the agonist-induced ROS generation as well as the bFGF-induced migration. Agonist-induced migration was also attenuated in SMC derived from Nox1 y/- mice and transduction of Nox1 restored normal migration. Likewise, SMC outgrowth in response to bFGF was attenuated in aortic segments from Nox1 y/- mice as compared with Nox1 y/+ mice. bFGF activated JNK but not Src in a Nox1-dependent manner. Consequently, phosphorylation of the adaptor protein paxillin, which is central for migration and secretion of matrix-metalloproteinases, were dependent on Nox1 as well as JNK but not Src. CONCLUSIONS: These data demonstrate that bFGF activates the Nox1-containing NADPH oxidase and that bFGF through a pathway involving ROS and JNK stimulates SMC migration.


Assuntos
Movimento Celular/fisiologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , NADPH Oxidases/metabolismo , Animais , Aorta/citologia , Células Cultivadas , Camundongos , Camundongos Endogâmicos , Modelos Animais , NADPH Oxidases/análise , Probabilidade , Distribuição Aleatória , Ratos , Ratos Endogâmicos , Espécies Reativas de Oxigênio/metabolismo , Sensibilidade e Especificidade , Transfecção
7.
J Immunol ; 175(4): 2132-43, 2005 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-16081779

RESUMO

UV irradiation is carcinogenic and immunosuppressive. Previous studies indicate that UV-mediated alteration of APCs and induction of suppressor T cells play a critical role in UV-induced immune suppression. In this study, we show that UV irradiation can directly (independently of APCs and suppressor T cells) inhibit T cell activation by blocking TCR-mediated phosphorylation of ERK and IkappaB via overactivation of the p38 and JNK pathways. These events lead to the down-modulation of c-Jun, c-Fos, Egr-1, and NF-kappaB transcription factors and thereby inhibit production of cytokines, e.g., IL-2, IL-4, IFN-gamma, and TNF-alpha, upon TCR stimulation. We also show that UV irradiation can suppress preactivated T cells, indicating that UV irradiation does not only impair T cell function in response to T cell activation, but can also have systemic effects that influence ongoing immune responses. Thus, our data provide an additional mechanism by which UV irradiation directly suppresses immune responses.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Terapia de Imunossupressão , Ativação Linfocitária/efeitos da radiação , NF-kappa B/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Transdução de Sinais/efeitos da radiação , Linfócitos T/efeitos da radiação , Raios Ultravioleta , Citocinas/antagonistas & inibidores , Citocinas/biossíntese , Citocinas/efeitos da radiação , Relação Dose-Resposta à Radiação , Ativação Enzimática/efeitos da radiação , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/efeitos da radiação , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/efeitos da radiação , Células Jurkat , Ativação Linfocitária/imunologia , NF-kappa B/biossíntese , NF-kappa B/fisiologia , NF-kappa B/efeitos da radiação , Proteínas Proto-Oncogênicas c-jun/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-jun/biossíntese , Proteínas Proto-Oncogênicas c-jun/efeitos da radiação , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores de Antígenos de Linfócitos T/efeitos da radiação , Transdução de Sinais/imunologia , Linfócitos T/enzimologia , Linfócitos T/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos da radiação
8.
J Immunol ; 174(11): 7075-84, 2005 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15905551

RESUMO

Aglaia (family Meliaceae) plants are used in traditional medicine (e.g., in Vietnam) for the treatment of inflammatory skin diseases and allergic inflammatory disorders such as asthma. Inflammatory diseases arise from inappropriate activation of the immune system, leading to abnormal expression of genes encoding inflammatory cytokines and tissue-destructive enzymes. The active compounds isolated from these plants are derivatives of rocaglamide. In this study we show that rocaglamides are potent immunosuppressive phytochemicals that suppress IFN-gamma, TNF-alpha, IL-2, and IL-4 production in peripheral blood T cells at nanomolar concentrations. We demonstrate that rocaglamides inhibit cytokine gene expression at the transcriptional level. At the doses that inhibit cytokine production, they selectively block NF-AT activity without impairing NF-kappaB and AP-1. We also show that inhibition of NF-AT activation by rocaglamide is mediated by strong activation of JNK and p38 kinases. Our study suggests that rocaglamide derivatives may serve as a new source of NF-AT-specific inhibitors for the treatment of certain inflammatory diseases.


Assuntos
Aglaia/química , Benzofuranos/farmacologia , Proteínas de Ligação a DNA/metabolismo , Imunossupressores/farmacologia , Proteínas Nucleares/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Benzofuranos/química , Benzofuranos/isolamento & purificação , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Citocinas/antagonistas & inibidores , Citocinas/biossíntese , Citocinas/sangue , Citocinas/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Células Jurkat , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/imunologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Fatores de Transcrição NFATC , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-jun/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-jun/biossíntese , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Linfócitos T/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia
9.
Eur J Immunol ; 34(4): 1111-8, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15048722

RESUMO

IL-4 plays a pivotal role in the development of the Th2 cell mediated humoral immune response and causes IgE-dependent allergic inflammatory diseases. Expression of IL-4 in differentiated Th2 cells is regulated by transcription factors such as NF-AT, AP-1 and NF-IL6. Recently, increasing evidence indicates that the pro-inflammatory transcription factor NF-kappa B may also participate inIL-4 expression. In this study, we show that the IL-4 promoter is synergistically activated by NF-kappa B, NF-AT and NF-IL6 at the NF-kappa B/NF-AT/NF-IL6 composite sites. In addition, we performed the chromatin immunoprecipitation technique to determine the functional relevance of NF-kappa B in the activation of the IL-4 gene in vivo. We demonstrate that NF-kappa B binds to the IL-4 promoter in vivo upon T cell activation. Inhibition of NF-kappa B nuclear translocation in living cells blocked binding of NF-kappa B to the IL-4 promoter. The data provide first evidence that NF-kappa B is directly involved in IL-4 transcription.


Assuntos
Regulação da Expressão Gênica , Interleucina-4/genética , Ativação Linfocitária/imunologia , NF-kappa B/imunologia , Células Th2/imunologia , Fatores de Transcrição/imunologia , Humanos , Interleucina-4/imunologia , Células Jurkat , Testes de Precipitina , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Transfecção
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